Volumetric mass density measurements of mesenchymal stem cells in suspension using a density meter

Summary To use regeneratively active cells for cell therapeutic applications, the cells must be isolated from their resident tissues. Different isolation procedures subject these cells to varying degrees of mechanical strain, which can affect the yield of cell number and viability. Knowledge of cell volumetric mass density is important for experimental and numerical optimization of these procedures. Although methods for measuring cell volumetric mass density already exist, they either consume much time and cell material or require a special setup. Therefore, we developed a user-friendly method that is based on the use of readily available instrumentation. The newly developed method is predicated on the linear relationship between the volumetric mass density of the cell suspension and the volumetric mass density, number, and diameter of the cells in the suspension. We used this method to determine the volumetric mass density of mesenchymal stem cells (MSCs) and compared it to results from the established density centrifugation.


INTRODUCTION
Mesenchymal stem/stromal cells (MSCs) are increasingly used in cell therapeutic applications because of their regenerative potential. 1,2 To enable clinical use, the cells can be isolated from their resident tissues (e.g., adipose tissue or bone marrow) by a broad variety of technical procedures, which can be of manual or automated nature. 3,4 The cell isolation procedures include processes such as agitation, filtration, and centrifugation that put mechanical strain on the cells. The cell media used to investigate the resistance of cells to mechanical shear and strain in an experimental setting need to be adjusted for the volumetric mass density of the cells. 5 Furthermore, the technical procedures mentioned above can be investigated using computational fluid dynamics (CFD) simulations to gain additional insights and reduce development time and costs compared to trial-and-error experiments. They allow the investigation of mechanical strain and mechanical forces and can predict the trajectory of individual cells in cell suspensions using particle models based on the Euler-Lagrange approach. [6][7][8] These models also require the volumetric mass density of the cells as an input parameter. Lastly, Grover et al. 9 report changes in cell volumetric mass density during biological processes such as cell cycle progression, apoptosis, differentiation, and certain diseases.
Differences between cell volumetric mass densities were originally utilized for the separation of different cell types via density gradient centrifugation, e.g., for the separation of different blood cell types (reviewed in Pertoft 10 ). Since then, multiple liquid media have been established for this technique; 11 among them are Ficoll 12 and Percoll. 13 Density gradient centrifugation has also been used to evaluate the cell density of human cells, 14 freshwater phytoplankters, 15 and polymer nanoparticles 16 quantitatively. This technique is material and time consuming, and due to the multiple manual steps that must be performed, it is prone to human error.
Suspended microchannel resonators (SMRs) have also been used in recent years for the determination of mass and volumetric mass density of biomolecules. 17,18 They facilitate the measurement of the volumetric mass density of individual cells, particles, or many single cells in succession 9,19-21 using yeast cells, transfused or malaria-infected human erythrocytes, human lung cancer cells, and mouse lymphoblasts. The SMR's working principle is similar to that of the density meter used in this study, only downscaled to the micrometer range. It consists of an oscillating microcantilever with a microchannel through which fluid and cells are flown. A single cell in the fluid changes the mass and resonance frequency of the oscillating cantilever. The volumetric mass density can then be calculated from these changes. However, since the microcantilever works under a vacuum for increased signal quality, the original manufacturing process of the measuring equipment is complex, but advances in the fabrication of SMRs have been reported. 22 Still, the technology is patented and currently only commercially available for particle or cell diameters below 5 mm (Malvern Panalytical). Also, optically induced electrokinetics has been used in a microfluidic chip to lift single cells and track the sedimentation via microscope and camera. 23 This technique also has an easier fabrication process than SMRs. In other experiments, glass or silica capillaries have been stretched to decrease their diameter, measure the resonance frequency, and thereby determine the buoyant mass. 24,25 Another approach is measuring the drag force of a single cell in a microfluidic channel on a lab-on-chip microfluidics system together with a syringe pump and an inverted microscope. 26 The study has been performed using yeast cells. However, there are currently no commercially available systems using all these approaches either.
Because the available techniques for determining the volumetric mass density of cells are either complex and time consuming or not widely commercially available, we established a user-friendly method for these cells using commercially available hardware such as density meters and cell counters. Both devices are already available in many laboratories.
Within this study, we refer to this method as cell suspension density measurement since it is key to measuring the volumetric mass density of the cell suspension which is increased by cells with higher volumetric mass density. In addition to measuring the volumetric mass density of the cell suspension, the volumetric mass density of the suspension medium, as well as the number of cells and the average cell diameter, has to be measured as accurately as possible. To evaluate its validity, we compared this novel method to the classic approach of measuring cell volumetric mass density via density centrifugation. For this study, we used primary human MSC from adipose tissue (adMSC).

Cell suspension density measurement: Theoretical approach
Because the volumetric mass density of the cell suspension and the suspension medium can be measured via density meter and the number of cells in the cell suspension and their average diameter can be measured via cell counter, the average volumetric mass density of the suspended cells can be calculated as follows: The mass of a cell suspension m susp is added up from the mass of the medium m medium (continuous phase) and the mass of the dispersed phase m cells (Equation 1).

m cells = r cells V cells (Equation 4)
Being able to measure the volumetric mass densities r susp and r medium and specify the volumes V susp , V medium , and V cells , one can calculate the volumetric mass density of the cells that are dispersed in the continuous fluid phase.
While V susp is the sample volume for the density measurement, the total volume of all cells in the sample of the cell suspension (total cellular volume) The diameter d cells;avg is volume-averaged and calculated from the cell diameter distribution with the number of cells n d at a diameter d in a range from d min to d max . The medium volume is then simply the difference between suspension volume and total cell volume.

RESULTS
Cell suspension density measurement was used to determine the volumetric mass density of cultured human adMSC isolated from the tissue of 6 different donors. For comparison, the volumetric mass density of the same cell material was measured using the conventional method of density centrifugation.
Cell suspension density measurement: Determination of the cell volumetric mass density using a density meter The calculated volumetric mass density of the adMSC in the cell suspension had a median of 1.0525 g/cm 3 (Table 1, Figure 1C). It was not normally distributed and varied between 1.042 and 1.056 g/cm 3 which was less than 1% fluctuation from the median. While the range was 0.014 g/cm 3 over all measurements of all six donors, it was slightly smaller with 0.001 g/cm 3 to 0.005 g/cm 3 for an individual donor. Note that donor 4 had marginally lower cell volumetric mass densities than the other donors.
The determined adMSC volumetric mass density ( The volumetric mass density of the cell suspension was therefore dependent on the cell number and, according to Equation 6, also on the total cellular volume in the cell suspension. It increased linearly with cell number (with R 2 of the linear regression being 0.835) and also with the total cellular volume V cells (R 2 of the linear regression 0.955). The linear fits not only matched well with the underlying data but also matched with the volumetric mass density of the DPBS solution ( Figure 1A at zero cell number, Figure 1B at zero total cellular volume).

Determination of the cell volumetric mass density using density centrifugation
The results in this section were determined using the commonly practiced density centrifugation and were intended to validate the volumetric mass density of adMSC that was obtained by using the proposed cell suspension density measurement.
The results in this section are presented as normalized cell numbers that are present in the different DPBS-LSM solutions with their varying density after the centrifugation. The DPBS-LSM solutions are mixtures of DPBS and Lonza Lymphocyte Separation Medium (LSM) (Lonza Group AG, Switzerland). In Figure 2A, the normalized total number of cells is plotted over the volumetric mass density of the different DPBS-LSM mixtures. All cell numbers are shown normalized, meaning relative to the number of cells initially filled into the 2 mL cell suspension before centrifugation. With an increased volumetric mass density of the DPBS-LSM mixture, fewer cells sedimented into the pellet during the centrifugation because the forces leading to sedimentation become smaller with decreasing density difference between the adMSC and DPBS-LSM mixture.
From 1.040 to 1.045 g/cm 3 , the cell number in the pellet decreased rapidly, while at the same time the cell number in the supernatant increased. At this DPBS-LSM volumetric mass density, cells were not able to sink through the DPBS-LSM mixture anymore. At DPBS-LSM volumetric mass densities higher than 1.045 g/cm 3 , the normalized total cell number in the pellet was close to 0% while the normalized total cell number in the supernatant was close to 100%. The magnitude of change in the normalized total cell number at different DPBS-LSM mixture densities indicates the actual volumetric mass density of the adMSC. Therefore, the ll OPEN ACCESS iScience 26, 105796, January 20, 2023 iScience Article change of the normalized total cell numbers is calculated using Equations 10 and 11 and plotted over the density of the DPBS-LSM solution ( Figure 2B). Figure Table S1.
The methodology used for cell suspension density measurement ( Figure 3) and density centrifugation (Figures 4 and 5) is described in the STAR Methods section of this paper.

Evaluation of the results
In this study, we determined the volumetric mass density of adMSC by measuring the volumetric mass density of cell suspension and cell medium with a density meter. We refer to this procedure as cell suspension density measurement. For comparison, we also performed density centrifugation measurements (a simplified version of the well-established density gradient centrifugation) on the same patient's material. We determined a median adMSC volumetric mass density of 1.0525 g/cm 3 using our method and 1.045 g/cm 3 (both for pellet and supernatant) using density centrifugation. We interpret this slight difference in volumetric mass density as the fact that nonviable cells and cell debris with their higher density 27 We are not aware of any previously published measurements of the adMSC volumetric mass density. However, the volumetric mass density of related cell or tissue material has been determined previously. For fibroblasts, a volumetric mass density of 1.03 g/cm 3 to 1.05 g/cm 3 has been determined. 29,30 For skeletal muscle tissue, a volumetric mass density of 1.06 g/cm 3 has been measured. 31,32 For fat cells (adipocytes) that differentiate from adMSC but contain a lipid vacuole, a volumetric mass density of 0.92 g/cm 3 has been determined. 33 The above-mentioned volumetric mass densities of fibroblasts were determined using density centrifugation while the volumetric mass density of skeletal muscle and adipose tissue was simply calculated from their dimensions and weight.  , if the cells have a lower or equal volumetric mass density. This approach significantly reduces the required precision, and thus reduces the susceptibility to errors in multistage pipetting procedures, as is common with standard density gradient centrifugation and the subsequent necessary processing steps. Density centrifugation has further limitations: because it only allows the determination of the cell volumetric mass density by determining the (maximum) change of cell number for DPBS-LSM solutions with different densities, it only provides approximate results. It is therefore less precise than the newly presented method, which is clearly reflected in the more differentiated results of cell suspension density measurements ( Figure 1C) compared to density centrifugation ( Figure 2B). Furthermore, the resolution depends on the step size of the mixed DPBS-LSM solutions. At the same time, it takes much longer to perform the density and cell number measurements for every individual DPBS-LSM solution. Therefore, substantially more cellular material is needed, thus limiting the number of measurements that can be performed. It does not take into account cell debris and therefore shows slightly lower cell volumetric mass densities compared to the volumetric mass density measurements of cell suspension and medium. It is ultimately also based on a density meter and a cell counter and thus reliant on the same hardware as our proposed method of cell suspension density measurement.
On the other hand, the resolution of the volumetric mass density measurements of cell suspension and medium depends on the accuracy of the density meter and the cell counter and not on the method itself. Therefore, both the accuracy of the proposed method and its sensitivity are discussed here.

Accuracy of the proposed method
The DSA 5000 M density meter has an accuracy 34 of 0.000007 g/cm 3 and a repeatability of 0.000001 g/cm 3 .
The NC-200 cell counter has a theoretical coefficient of variation (CV) of 2.7% at a cell concentration of 1 3 10 6 cells/mL compared to CVs of more than 10% for manual counting in hemocytometers. 35 Preparation and handling of the cell suspension samples are crucial for the proposed method, as is the accuracy of the measurement systems. The exact number of cells in the sample and the exact volume of the cell suspension sample V susp are essential for the calculation of the cell volumetric mass density r cells via Equation 5. The cell suspension sample is therefore prepared using microliter pipettes with high accuracy. The accuracy of the scale (0.1 mL) of the 2.0 mL inlet syringe is insignificant for its subsequent filling with the cell suspension sample since the cell suspension is not completely loaded into the density meter and a part of the cell suspension sample remains in the syringe. It is important to do the measurements with the density meter directly after preparing the cell suspension sample. Otherwise, the cells will sediment and aggregate which may affect the density measurement. Air bubbles that can impair the density measurements are easily visible on the monitor of the DSA 5000 M density meter and can therefore be removed from the measurement glass tube with the help of the 0.5 mL extra volume that has been filled into the syringe. It is also noteworthy that the measured volumetric mass density of the DPBS medium has not been included in the regression but is touched by the regression line for a cell number or total cellular volume of zero. This shows the accuracy of the proposed method. Also, it confirms the accuracy of the density meter and cell counter which is (together with the precise handling of the samples) essential for an exact calculation of the cell volumetric mass density. For all six individuals, the volumetric mass density fluctuates only at the third decimal place while the first two decimal places are stable (Table 1). This demonstrates that our method even allows us to distinguish between individuals. Also, the good agreement with the results from density centrifugation using cells from the same patients and finally the good agreement with the volumetric mass densities of similar cell and tissue material from the literature indicate that the proposed method can accurately and reproducibly determine the volumetric mass density of a given cell type.  iScience Article With this method, it is possible to adjust the volumetric mass density of cell media, e.g., to investigate the resistance of cells to mechanical shear and strain or to perform numerical flow simulations. This is consistent with Grover et al. 9 who show that there are slight differences in volumetric mass density for other cell types such as erythrocytes or L1210 mouse lymphocytic leukemia cells. Nevertheless, it remains a challenge to distinguish these donor-specific differences in cell volumetric mass density from measurement inaccuracies. However, with the precautions mentioned at the beginning of this section regarding the handling of the cell suspension and cleaning of the measurement glass tube before use (using 99.7% ethanol), it is possible to reduce the fluctuations of the measurements significantly enough to determine these donor-specific differences. Increasing the number of donors as well as the number of cell samples per donor could also increase the accuracy of the determinations of the volumetric mass density of a certain cell type and the donor-specific differences. Although three technical replicates per cell sample seem to be sufficient, this number could be increased to improve accuracy. With our proposed method, it could be further investigated as to whether there is a correlation between the difference in cell volumetric mass density and other cellular properties or pathologies, as outlined by Grover et al. 9 In the context of density centrifugation, it is noteworthy that the viability of the few cells that passed through the DPBS-LSM solutions with densities higher than 1.040 g/cm 3 and ended up in the pellet decreased by about 30%. We believe that this effect is due to the increased viscosity and resulting higher shear forces of the DPBS-LSM solution at higher volumetric mass densities. This phenomenon could underline the fact that the volumetric mass density and viscosity of cell media are important parameters for procedures where cell suspensions are handled, especially if they experience high shear. It consequently underlines the need for density-adjusted cell media.

Sensitivity of the proposed method
We have developed a new method for determining the volumetric mass density of cells in suspension. This method is based on measuring the volumetric mass density of a cell suspension and its pure cell medium with a density meter and counting the cells in the suspension and then calculating the volumetric mass density of the cells in the cell suspension. We tested this new method with human adMSC and found that the volumetric mass density determined was consistent with the volumetric mass density of the same patient material determined by density centrifugation. Thus, the method we developed shows more precise results, since the measurement resolution depends only on the precision of the density meter and not on the number of mixed solutions (DPBS-LSM) or even a gradient. In addition, the new method requires less time and substantially fewer cells.

Limitations of the study
This study presents an approach to determine volumetric mass density of cells in suspension. However, there are some limitations to this study. This study has a small sample size, but this specific study is only an illustration of a use case and the results for the volumetric mass density are close to each other. Also, only female donors were available for this study. It would be interesting to also include male donors in future studies to see if the volumetric mass density of cells is dependent on gender.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: The isolation of the cells from human adipose tissue has been previously described. 36 After isolation, the cells were cultured in tissue culture flasks (Greiner Bio-One GmbH, Austria). The cell culture medium consisted of Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific Inc., USA) supplemented with 1% penicillin/streptomycin (P/S, Thermo Fisher Scientific Inc., USA) and 10% fetal calf serum (FCS, PAN-Biotech GmbH, Germany).
After 24 h of adherence, cells positive for the surface marker CD34 were separated from other plasticadherent cells using a magnetic bead selection system (Thermo Fisher Scientific Inc., USA). The cells thus obtained were further cultivated in tissue culture flasks. When they reached confluence after about 5 days, the cells were detached from the flask with 0.25% trypsin-EDTA (Thermo Fisher Scientific Inc., USA). They were divided in a 1:3 surface ratio onto new tissue culture flasks. This step was repeated after another 5 days. When the cells on the resulting flasks had then reached confluence, they were used for the two methods of cell volumetric mass density determination. The cells were detached from the culturing flasks with 0.25% trypsin-EDTA and were transferred into Dulbecco's PBS (DPBS, PAN-Biotech GmbH, Germany) with 10% FCS. After centrifugation (5 min, 400 x g, room temperature), the cells were transferred into culture medium. The few potentially remaining magnetic beads from the CD34 selection were removed with a magnet. The adMSC were then stored on ice for further use.

Cells suspension density measurement: Density meter
Prior to the measurements in the DSA 5000 M density meter (Anton Paar GmbH, Austria) the cell number and cell viability in the cell suspension were determined with the NucleoCounter NC-200 cell counter (ChemoMetec A/S, Denmark). The cell suspension was then divided into three fractions containing three different cell concentrations suitable for providing a reliable signal in the density meter. The range of suitable cell concentrations ('cell samples') had been determined in preliminary testing. Each of the three fractions were centrifuged (5 min, 400 x g, room temperature) and suspended in 3 mL DPBS. Of those 3 mL, 500 mL were used for another measurement of the cell number, cell viability, and cell diameter distribution with the NC-200. All three parameters were measured in technical triplicates of the same sample. The results of these measurements are displayed as average values of the technical triplicate measurements. The remaining 2.5 mL of the suspension were used for the measurement of the volumetric mass density in the DSA 5000 M density meter. In addition to the volumetric mass density of the cell suspension r susp the volumetric mass density of the suspension medium (DPBS), r medium , was measured using the density meter.
The DSA 5000 M contains an oscillating U-shaped borosilicate glass tube filled with the fluid or suspension (Figures 3 and S1A). The resonance frequency of the oscillation of the U-shaped glass tube and therefore its period T U is dependent on the volumetric mass density of the sample T U and T ref are the period of the U-shaped glass tube and the period of a reference oscillation while A and B are adjustment constants. f 1 and f 2 are correction functions for temperature and viscosity. 34 A 2.0 mL syringe (B. Braun SE, Germany) was filled with V susp = 2:5 ml and was connected to the inlet of the density meter via a standard Luer adapter. The plunger was removed from a 1.0 mL syringe (B. Braun SE, Germany) and the barrel was then connected to the outlet of the density meter via a standard Luer adapter and served as a capillary. This setup allowed for reducing the sample volume to 2.5 mL but leaving room to pull or push out possible air bubbles ( Figure S1B) during the filling of the system. The sample was cooled down to the target temperature of 20.0 C via the built-in Peltier element. This took about 5 min per sample.
To avoid sedimentation and aggregation of cells the measurements had to be undertaken as quickly as possible. To avoid cellular residues in the measurement chamber that may affect the following measurements, after each measurement the glass tube was cleaned with distilled water and undenatured ethanol >99.7%. Lastly, the glass tube was dried with an air stream. Because the average diameter of the NC-200 standard output was calculated as an arithmetic average, it was not consistent with the total cellular volume and their specific diameter. Since the cell diameter distribution could be extracted from the NC-200 raw data, the volume-averaged cell diameter was exported and calculated. It must be noted that the diameter distribution from the NC-200 was not exported as in cells/ml but in actual cell events from the measurement window. Therefore, in Equation 7 P n d the sum of all cells of all diameters in the cell counting sample was not equal to n cells which comes also from the cell counter but is projected to the 2.5 mL density measurement sample.
The NC-200 dataset was loaded into the NC-3000 software (ChemoMetec A/S, Denmark) where the dataset was exported in the fluorescence-activated cell sorting (FACS) data format. The data were then loaded into the FACS analysis software FlowJo v10 (Flowjo, LLC, USA) and the diameter distribution was exported from the histogram using the Windows Clipboard (Microsoft Corporation, USA). The diameter distribution could then be imported and the volume-averaged cell diameter d cells;avg could be calculated in any spreadsheet software, e.g., Excel (Microsoft Corporation, USA) via Equation 7. For adMSC diameters between d min = 1 mm and d max = 41 mm were included in the calculation of the volume-averaged cell diameter.

Cell volumetric mass density: Density centrifugation
To validate the results from cell suspension density measurements, a second method was performed: density centrifugation.
After measuring the cell number of the cell suspension (see section experimental model and subject details) using the NC-200 a portion of the cell suspension containing 20 x 10 6 cells was centrifuged (5 min, 400 x g, room temperature) and suspended in 21 mL DPBS. 500 mL were used for cell number and cell diameter measurement with the NC-200 and 20 mL of the suspension were divided into 2 mL aliquots placed over different media with different volumetric mass densities to determine the cell's volumetric mass density via density centrifugation. These media were mixtures of DPBS and Lonza Lymphocyte Separation Medium (LSM) (Lonza Group AG, Switzerland). LSM itself is a mixture of sodium diatrizoate (Hypaque) and Ficoll and has a density of 1.077 g/cm 3 while DPBS has a density of 1.0052 g/cm 3 . DPBS-LSM mixtures with the volumetric mass densities of 1.010, 1.015, 1.020, 1.025, 1.030, 1.035, 1.040, 1.045, 1.050 and 1.055 g/cm 3 were prepared.
After the preparation of the DPBS-LSM mixtures, the volumetric mass densities of the DPBS-LSM mixtures were verified using the DSA 5000 M. In addition, the dynamic viscosities, osmolalities, and the pH of the DPBS-LSM mixtures were determined to ensure cell-friendly conditions such as an osmolality in the isosmotic range between 275 and 290 mOsmol/kg and a pH around 7 over the entire mixture range from 100% DPBS to 100% LSM. The dynamic viscosity was measured using a Haake MARS II rotational rheometer (Thermo Fisher Scientific Inc., USA), the osmolality using an Osmomat 3000 (Gonotec GmbH, Germany), and the pH using a Seven Compact (Mettler Toledo Inc., USA). Both volumetric mass density and dynamic viscosity increased with increasing volume fraction of LSM in the DPBS-LSM solution. The fluid properties of the DPBS-LSM mixture are illustrated in Figure 4. The 2 mL of the cell suspension were carefully layered over 9 mL of DPBS-LSM mixture with one of the 10 defined volumetric mass densities ( Figure 5, left) in a 50 mL centrifugation tube (Greiner Bio-One GmbH, Austria). The tube was centrifuged for 5 min at 400 x g at room temperature. The cell numbers in the complete supernatant (consisting of DPBS and the DPBS-LSM mixture with parts of the cells) and in the pellet were determined using the NucleoCounter NC-200.
To interpret the density centrifugation data, the change (first derivative) of the normalized total cell number in the pellet (blue) and the supernatant (red) was plotted against the volumetric mass density of the DPBS-LSM mixture. The change in normalized total cell number at a given DPBS-LSM volumetric mass density was calculated from both neighboring points using a central differencing scheme as in Equations 10 and 11.

QUANTIFICATION AND STATISTICAL ANALYSIS
The details for all statistical analyses are provided in the table and figure legends. The statistical analyses were performed using MATLAB (The Mathworks Inc., USA). n = 6 represents the number of donors used in this research, for both cell suspension density measurement using the density meter as well as for density centrifugation.Performing cell suspension density measurement, triplicate samples from each donor were used. Cell counting was also done with technical triplicates for each donor sample. The technical replicates were averaged using the arithmetic mean. The cell diameters have been volume-averaged from the cell counter's diameter distribution according to Equation 7 for every technical replicate and then the arithmetic mean of all 3 technical replicates of all 3 samples from a donor has been calculated. The median volumetric mass density of all 3 replicates of all 6 donors is used to represent the adMSC volumetric mass density determined from the cell suspension density measurement. Variances of the median have been judged in terms of % deviation. Variances between samples of a donor and variances between donors have also been compared in terms of the range. To further judge the validity of the theoretical approach as well as the accuracy of the measurements, linear regressions have been performed for the relationship between the measured cell suspension volumetric mass density and both cell number as well as total cellular volume using MATLAB. The coefficient of determination R 2 has been computed for both regressions. For density centrifugation, cell counting of the supernatants and pellets of all six donors was done in technical triplicates. The technical replicates were averaged using the arithmetic mean. The median of the normalized cell numbers of all technical replicates for all 6 donors was calculated for both supernatant and pellet of every DPBS-LSM mixture with respective density. The adMSC volumetric mass density was then derived from the maximum change of normalized cell number using a central differencing scheme from the median of both supernatant and pellet.